N-(3,3-Dimethylbutyl)-L-aspartyl-D-α-aminoalkanoic acid N-(S)-1-phenyl-1-alkanamide useful as a sweetening agent

ABSTRACT

The present invention has as subject compounds of formula: ##STR1## in which Y is C 2  H 5 , (CH 3 ) 2  CH or (R) CH(OH)CH 3 , and R&#34; is (S) CH(C 2  H 5 )C 6  H 5 , (S) CH(CH 3 )C 6  H 5  or (R) CH(CH 2  OCH 3 )C 6  H 5 . These compounds are useful as sweetening agents.

The present invention relates to novel compounds derived fromdipeptides, useful as sweetening agents, as well as to their method ofpreparation.

These novel compounds are particularly useful for sweetening a varietyof products, especially drinks, foods, confectionery, pastries, chewinggums, hygiene products and toiletries, as well as cosmetic,pharmaceutical and veterinary products.

It is known that, in order to be usable on an industrial scale, asweetening agent must possess firstly an intense sweetening potency,making it possible to limit the cost of use, and secondly a satisfactorystability, i. e. a stability compatible with the conditions of use.

Amongst the sweetening agents currently on the market, a dipeptidederivative L-aspartyl-L-phenylalanine methyl ester, known under the nameof aspartame and having the following formula: ##STR2## is the mostwidely used (U.S. Pat. No. 3,475,403). The relatively weak sweeteningpotency of this compound is about 120 to 180 times that of sucrose on aweight basis. Despite its excellent organoleptic properties, the maindisadvantage of this compound is that it is an expensive product onaccount of its relatively low sweetening intensity, and that it has arather low stability under the usual conditions of use of sweeteningagents, limiting its areas of industrial applications.

In the FR 92 13615 document, the Applicants have proposed sweeteningagents of the following general formula: ##STR3## in which: R is asaturated or non-saturated, acyclic, cyclic or mixed hydrocarbon grouphaving from four to thirteen carbon atoms;

n is equal to 1 or 2; and

R' is represented by the following formula: ##STR4## in which: Y isselected from the groups COOCH₃, COOC₂ H₅, CH₃, CH₂ OH, CON(CH₃)₂, C₆H₅, 2-furyl, and H;

Z is selected from the groups CH₂ C₆ H₅, C₆ H₅, n-C₄ H₉, COOCH₃, COOC₂H₅, COOC₃ H₇, COOfenchyl, and CONHR" in which R" is selected from thegroups CH₃, CH₂ CH₃, CH₂ CH₂ CH₃, CH₂ CH₂ CH(CH₃)₂, CH(CH₃)COOCH₃,CH(c-C₃ H₅)₂, CH(c-C₃ H₅)C(CH₃)₃, fenchyl, 2,6-dimethylcyclohexyl,2,2,5,5-tetramethylcyclopentyl, and 2,2,4,4-tetramethyl-3-thietanyl.

One form of implementation described in the FR 92 13615 document coversthe L-aspartic acid derivatives (n=1) of the following formula: ##STR5##in which R, Y and R" are such as previously defined.

A preferred compound in this form of implementation of the invention isN-(3,3-dimethylbutyl)-L-aspartyl-N-(dicyclopropylcarbinyl)-D-alaninamideof formula: ##STR6## in which Y represents the methyl group and R"represents the dicyclopropylcarbinyl group which possesses a sweeteningpotency of 2,500 times that of sucrose on a weight basis with respect toa 2% sucrose solution.

It has been observed, and this constitutes the foundation of theinvention, that it was possible to obtain novel sweetening compounds ofthe precited formula for suitably selected substituents Y and R",different from those provided in this prior document. Thus, when Yrepresents an ethyl, isopropyl or (R)-α-hydroxyethyl group and R"represents an (S)-α-ethylbenzyl, (S)-α-methylbenzyl or(R)-α-methoxymethylbenzyl group, novel compounds are obtained whichpossess excellent organoleptic qualities associated with a very highsweetening potency, up to 8,000 times the sweetening potency of sucroseon a weight basis. Furthermore, the stability of these compounds inacidic or neutral solution is distinctly higher than that of aspartame,which should have as effect to enlargen, with respect to aspartame,their possibilities of use in the food preparations.

The present invention has therefore as aim to provide novel sweeteningagents responding to the following formula: ##STR7## in which: Y is C₂H₅, or CH(CH₃)₂ or (R) CH(OH)CH₃ ; and

R" is (S) CH(C₂ H₅)C₆ H₅, (S) CH(CH₃)C₆ H₅ or (R) CH(CH₂ OCH₃)C₆ H₅.

The compounds responding to a particularly advantageous form ofimplementation of the invention are theN-(3,3-dimethylbutyl)-L-aspartyl-D-α-aminobutyric acidN-(S)-1-phenyl-1-propanamide of formula: ##STR8## which has a sweeteningpotency of about 8,000 times that of sucrose on a weight basis withrespect to a 2% sucrose solution, theN-(3,3-dimethylbutyl)-L-aspartyl-D-valine N-(S)-1-phenyl-1-propanamideof formula: ##STR9## which has a sweetening potency of about 3,000 timesthat of sucrose on a weight basis with respect to a 2% sucrose solution;

the N-(3,3-dimethylbutyl)-L-aspartyl-D-α-aminobutyric acidN-(S)-1-phenyl-2-methoxy-1-ethanamide of formula: ##STR10## which has asweetening potency of about 4,000 times that of sucrose on a weightbasis with respect to a 2% sucrose solution; and

the N-(3,3-dimethylbutyl)-L-aspartyl-D-valineN-(S)-1-phenyl-2-methoxy-1-ethanamide of formula: ##STR11## which has asweetening potency of about 4,000 times that of sucrose on a weightbasis with respect to a 2% sucrose solution.

Moreover, it has been demonstrated that the stability of thesecharacteristic compounds of the invention is distinctly higher than thatof aspartame under the common conditions of use for the foodpreparations. This advantage is all the more important because one ofthe limits of the use of aspartame in certain food preparationsoriginates from its very low stability in media near to neutrality, i.e. for values of pH of about 7, values of pH which are frequentlyencountered in products such as dairy products, pastries or otherpreparations needing a high cooking temperature.

It has also been demonstrated that the stability of the compounds of theinvention is further improved in acidic media at pH values of about 3,which correspond to pH values of soft drinks which constitute one of themajor applications of sweetening agents.

Thus, by a study of accelerated ageing by prolonged heating an aqueoussolution at pH 3 at 70° C., it has been shown that one of thecharacteristic compounds of the invention, theN-(3,3-dimethylbutyl)-L-aspartyl-D-α-aminobutyric acidN-(S)-1-phenyl-1-propanamide, possesses a half-life of about 72 hours.By way of comparison, the half-life of aspartame under the sameconditions is only about 24 hours, which corresponds to a stabilityabout 3 times greater for the compound according to the invention.

A same study of accelerated ageing at pH 7 has shown that the samecompound, the N-(3,3-dimethylbutyl)-L-aspartyl-D-α-aminobutyric acidN-(S)-1-phenyl-1-propanamide, possesses a half-life of about 12 days,while the half-life of aspartame under the same conditions is only 10minutes, which corresponds to a stability about 1,700 times greater forthe compound according to the invention. Comparable results have beenobtained for the other characteristic compounds of the invention.

From the fact of their high sweetening potency, another advantage of thecompounds of the invention compared to aspartame is to allow, in theirapplication to food products, the use of very low quantities of activeagent. It is therefore for example that it is possible to replace, inone litre of soft drink, 550 mg of aspartame by about 20 mg of theN-(3,3-dimethylbutyl)-L-aspartyl-D-α-aminobutyric acidN-(S)-1-phenyl-1-propanamide of the invention, and to therefore reduceby about 27 times the quantities of sweetening agent consumed, always inmaintaining the identical organoleptic qualities.

The sweetening agents of the present invention may be added to anyedible product to which it is desired to give a sweet taste, providedthat it is added in sufficient proportions to attain the level ofsweetness desired. The optimal use concentration of the sweetening agentwill depend upon a variety of factors such as, for example, thesweetening potency of the sweetening agent, the conditions of storageand use of the products, the particular constituents of the products andthe level of sweetness desired. Any qualified person can easilydetermine the optimal proportion of sweetening agent which must beemployed in order to obtain an edible product by carrying out routinesensory analyses. The sweetening agents of the present invention is, ingeneral, added to the edible products in proportions, according to thesweetening potency of the compound, ranging from 5 mg to 50 mg ofsweetening agent per kilogramme or per litre of edible product. Theconcentrated products will obviously contain greater quantities ofsweetening agent, and will then be diluted following the finalintentions of use.

The sweetening agents of the present invention may be added in the pureform to products to be sweetened, but because of their high sweeteningpotency they are generally mixed with an appropriate carrier or bulkingagent.

Advantageously, the appropriate carriers or bulking agents are selectedfrom the group consisting of polydextrose, starch, maltodextrins,cellulose, methylcellulose, carboxymethylcellulose and other derivativesof cellulose, sodium alginate, pectins, gums, lactose, maltose, glucose,leucine, glycerol, mannitol, sorbitol, sodium bicarbonate, phosphoric,citric, tartaric, fumaric, benzoic, sorbic and propionic acids and theirsodium, potassium and calcium salts, and equivalents thereof.

The sweetening agent of the invention may, in an edible product, beemployed as the only sweetening agent, or in combination with othersweetening agents such as sucrose, corn syrup, fructose, sweet dipeptideanalogues or derivatives (aspartame, alitame), neohesperidindihydrochalcone, hydrogenated isomaltulose, stevioside, the L sugars,glycyrrhizin, xylitol, sorbitol, mannitol, acesulfame-K, saccharin andits sodium, potassium, ammonium and calcium salts, cyclamic acid and itssodium, potassium and calcium salts, sucralose, monellin, thaumatin andequivalents thereof.

The compounds of the present invention are prepared by a reductiveN-alkylation consisting in condensing the dipeptide precursor offormula: ##STR12## in which Y and R" are such as defined previously,with 3,3-dimethylbutyraldehyde in the presence of a reducing agent. Thisreducing agent is either hydrogen under a relative pressure comprisedbetween 1 and 3 bars and in the presence of a catalyst based on platinumor palladium following the procedure described in the WO 95/30689document, or sodium cyanoborohydride following the procedure describedin the FR 92 13615 document.

The dipeptide precursor of the above formula can easily be obtained bycarrying out the basic principles of peptide synthesis: protection ofthe amino and carboxyl groups of the amino acid precursors and theirdeprotection, and classical methods of activation and peptide coupling.

These techniques are described in a detailed fashion in numerouspublications, amongst which M. Bodanszky and A. Bodanszky, The Practiceof Peptide Synthesis, Springer-Verlag, New York, 1984, 284 pp. may mostparticularly be cited.

The purification of the compounds of the invention is performedaccording to the standard techniques such as recrystallization andchromatography. Their structure and their purity have been checked byclassical techniques (thin layer chromatography, high performance liquidchromatography, infrared spectroscopy, nuclear magnetic resonance,elementary analysis).

The sweetening potency of the compounds described in the examples hasbeen evaluated by a group of eight experienced people. For this, thecompound, in aqueous solution at varying concentrations, is compared,with respect to taste, to a control solution of sucrose of concentration2%, 5%, or 10%. The sweetening potency of the test compound comparedwith sucrose then corresponds to the weight ratio between the compoundand sucrose for equal sweetening intensity, i.e. when the sweet tastesof the solution of the test compound and the control solution of sucroseare considered by a majority of people to have the same sweeteningintensity.

The stability of the compounds of the invention and aspartame wasmeasured by determining, using high performance liquid chromatography(HPLC), the amount of product remaining after an accelerated ageing inacidic medium (phosphate buffer at pH 3) or in neutral medium (phosphatebuffer at pH 7) and at the temperature of 70° C. The stability of thecompound thus tested is evaluated by its half-life ( time correspondingto 50% degradation).

The manner with which the invention may be carried out and theadvantages which follow therefrom shall emerge better from the examplesof implementation which follow.

EXAMPLES

Preparation of N-(3,3-dimethylbutyl)-L-aspartyl-D-α-aminobutyric acidN-(S)-1-phenyl-1-propanamide of formula: ##STR13##

A mixture of 15 g (0.145 mole) of D-α-aminobutyric acid (Aldrich productNo. 11,612-2) and 5.82 g (0.145 mole) of sodium hydroxide in 150 cm³ ofwater is cooled to 0° C. To this solution are simultaneously added, in adropwise fashion, 24.38 g (0.145 mole) of benzyl chloroformate and 5.82g (0.145 mole) of sodium hydroxide in aqueous solution (4N). Stirring isthen maintained for 3 hours at 0° C. The reaction mixture is washed withdiethyl ether (3×30 cm³) and then acidified by a solution ofhydrochloric acid (6N) until a pH of about 1 is obtained. The whiteprecipitate formed is separated off by filtration, washed with waterthen dried. 26 g (yield 75%) of N-benzyloxycarbonyl-D-α-aminobutyricacid are finally obtained whose melting point is 80° C.

To a solution of 3 g (13 mmoles) of the compound thus prepared in 50 cm³of tetrahydrofuran cooled to -15° C. are successively added 1.28 g (13mmoles) of N-methylmorpholine and 1.15 g (13 mmoles) of isobutylchloroformate. After 2 minutes of stirring at this temperature, 1.71 g(13 mmoles) of (S)-1-phenyl-1-propanamine (previously prepared accordingto the document J. Chem. Soc., 1940, pp. 336-8). The reaction mixture isslowly warmed up and then stirred for 2 hours at room temperature. TheN-methylmorpholine hydrochloride precipitate is removed by filtration,then washed with 20 cm³ of tetrahydrofuran. The filtrates areconcentrated to dryness in vacuo and the residue obtained is taken upinto 150 cm³ of diethyl ether. The solution obtained is washedsuccessively with a 0.1N solution of hydrochloric acid, a 5% solution ofsodium carbonate then with water (3×30 cm³ for each washing). Afterdrying of the ethereal solution over anhydrous sodium sulfate, thesolution is concentrated to dryness in vacuo, which leads to 3.8 g of awhite solid residue, N-benzyloxycarbonyl-D-α-aminobutyric acidN-(S)-1-phenyl-1-propanamide (yield 82%) whose melting point is 129° C.Its purity is checked by thin layer chromatography on silica gel G 60(silica support Merck No. 1.05554), eluting with chloroform-acetone(9-1), and visualizing by the potassium dichromate-concentratedsulphuric acid mixture, Rf=0.45.

A solution of 3.6 g (10 mmoles) of N-benzyloxycarbonyl-D-α-aminobutyricacid N-(S)-1-phenyl-1-propanamide in 100 cm³ of methanol is submitted,in the presence of 350 mg of 10% palladium on activated charcoal (Flukaproduct No. 75990), to hydrogen under atmospheric pressure for 18 hours.After removal of the catalyst by filtration, the solution isconcentrated to dryness in vacuo. 2.1 g (yield 91%) of D-α-aminobutyricacid N-(S)-1-phenyl-1-propanamide, in the form of an oily residue, isobtained. Its purity is checked by thin layer chromatography on silicagel G 60 (silica support Merck No. 1.05554), eluting with butanol-aceticacid-water (8-2-2), and visualizing with ninhydrin, Rf=0.57.

To a solution of 3.58 g (10 mmoles) of N-benzyloxycarbonyl-L-asparticacid β-benzyl ester (Bachem product No. C-1350) in 50 cm³ oftetrahydrofuran cooled to -15° C. are successively added 1 g (10 mmoles)of N-methylmorpholine and 1.37 g (10 mmoles) of isobutyl chloroformate.After 2 minutes of stirring at this temperature, 2.1 g (10 mmoles) ofthe previously prepared D-α-aminobutyric acidN-(S)-1-phenyl-1-propanamide are added. The reaction mixture is slowlywarmed up and then stirred for 2 hours at room temperature. TheN-methylmorpholine hydrochloride precipitate is removed by filtration,and then is washed with 20 cm³ of tetrahydrofuran. The filtrates areconcentrated to dryness in vacuo and the residue obtained is trituratedin 50 cm³ of diethyl ether. The white solid formed is separated byfiltration and is then washed again with 20 cm³ of diethyl ether. 5 g(yield 89%) of N-benzyloxycarbonyl-β-benzylester-L-aspartyl-D-α-aminobutyric acid N-(S)-1-phenyl-1-propanamide arethus obtained whose melting point is 130° C. Its purity is checked bythin layer chromatography on silica gel G 60 (silica support Merck No.1.05554), eluting with chloroform-acetone (8-2), visualizing with thesulfochromic mixture, Rf=0.64.

A solution of 5 g (8.9 mmoles) of N-benzyloxycarbonyl-β-benzylester-L-aspartyl-D-α-aminobutyric acid N-(S)-1-phenyl-1-propanamide in100 cm³ of methanol is submitted, in the presence of 500 mg of 10%palladium on activated charcoal (Fluka product No. 75990), to hydrogenunder atmospheric pressure for 18 hours. After removal of the catalystby filtration, the solution is concentrated to dryness in vacuo. 2.4 g(yield 80%) of L-aspartyl-D-α-aminobutyric acidN-(S)-1-phenyl-1-propanamide are thus obtained in the form of a whitesolid. Its purity is checked by thin layer chromatography on silica gelG 60 (silica support Merck No. 1.05554), eluting with butanol-aceticacid-water (8-2-2), and visualizing with ninhydrin, Rf=0.50. Its meltingpoint in this amorphous state is 195° C.

In a reactor equipped with a stirrer assuring a very good transfer ofgaseous hydrogen into the liquid phase, are introduced, with stirring,in this order: 15 cm³ of a 0.1M aqueous solution of acetic acid, 26 mgof palladium on activated charcoal (Fluka Product No. 75990), 58 mg(0.57 mmole) of 3,3-dimethylbutyraldehyde of commercial origin (AldrichNo. 35,990-4), 15 cm³ of methanol and 130 mg (0.38 mmole) of thepreviously prepared L-aspartyl-D-α-aminobutyric acidN-(S)-1-phenyl-1-propanamide.

After having purged the reactor with a current of nitrogen gas, themixture is submitted to a hydrogenation at the relative pressure of 1-2bars (0.1-0.2 MPa) at room temperature for 18 hours, then again for 8hours after another addition of 19 mg (0.19 mmole) of3,3-dimethylbutyraldehyde. The progress of the reaction is monitored bythin layer chromatography (TLC) on Silica gel 60 F254 support onaluminium sheets (Merck No.1.05554), eluting with butanol-aceticacid-water (8:2:2), and visualizing with ninhydrin: Rf 0.67.

The reaction is finally interrupted by purging the reactor with acurrent of nitrogen gas and separating the catalyst by filtration on afine filter (0.5 μm). The solution is then concentrated by evaporationin vacuo and the white solid obtained is washed with about 50 cm³ ofdiethyl ether. 150 mg of theN-(3,3-dimethylbutyl)-L-aspartyl-D-α-aminobutyric acidN-(S)-1-phenyl-1-propanamide are finally obtained (yield 92%) in theform of a white powder of high purity (greater than 98% by H. P. L. C.)and whose melting point is 167° C.

Molecular formula: C₂₃ H₃₇ N₃ 0₄ NMR (200 MHz, 1H, ppm), DMSO D6: 0.80(s, 15H), 1.33 (t, 2H), 1.67 (m, 4H), 2.4 (m, 4H), 3.54 (m, 1H), 4.30(q, 1H), 4.70 (q, 1H), 7.22 (m, 1H), 7.28 (s, 5H), 8.35 (t, 2H).

High performance liquid chromatography on a Merck column of "Lichrospher100 RP-18 endcapped" type, 244 mm in length, 4.6 mm in diameter, eluent:65 mM ammonium acetate buffer--acetonitrile (70:30), flow: 1 ml/min,detector: refractometer, retention time: 16.1 min.

The sweetening potency of this compound corresponds approximately, on aweight basis, to 8,000 times that of sucrose by comparison with a 2%solution of sucrose, to 6,000 times that of sucrose by comparison with a5% solution of sucrose and to 5,000 times that of sucrose by comparisonwith a 10% solution of sucrose.

By comparison with aspartame, an aqueous solution of 20 mg/L of thiscompound is equivalent in sweetening potency to a solution of 550 mg/Lof aspartame, which corresponds to a sweetening potency about 27 timeshigher than that of aspartame.

Given in the annexed FIG. 1 is a comparative diagram of the stabilitycurves of aspartame (curve 1) and of theN-(3,3-dimethylbutyl)-L-aspartyl-D-α-aminobutyric acidN-(S)-1-phenyl-1-propanamide of the present invention (curve 2) obtainedduring an accelerated ageing by heating their solutions in acidic mediumat pH 3 (phosphate buffer) at 70° C. Under these conditions, thehalf-life of aspartame is 24 hours, while the half-life of the compoundof the invention is about 72 hours, which corresponds to a stabilitywhich is three times greater in favour of the compound of the inventionwith respect to aspartame.

Given in the annexed FIG. 2 is a comparative diagram of the stabilitycurves of aspartame (curve 1) and of theN-(3,3-dimethylbutyl)-L-aspartyl-D-α-aminobutyric acidN-(S)-1-phenyl-1-propanamide of the invention (curve 2) obtained duringan accelerated ageing by heating their solutions in neutral media of pH7 at 70° C. Under these conditions, aspartame is very little stable(half-life of 10 minutes) while the compound of the invention possessesa half-life of about 12 days, which corresponds to a stability which isabout 1,700 times higher than that of aspartame.

The sweetening potency of other compounds according to the invention,obtained following an experimental protocol similar to that describedabove and that the person skilled in the art will easily find, is givenin Table 1.

                  TABLE 1                                                         ______________________________________                                         ##STR14##                                                                    Y         R"             Sweetening potency*                                  ______________________________________                                        C.sub.2 H.sub.5                                                                         S (CH(C.sub.2 H.sub.5)C.sub.6 H.sub.5                                                        8,000                                                (CH.sub.3).sub.2 CH                                                                     (S) CH(C.sub.2 H.sub.5)C.sub.6 H.sub.5                                                       3,000                                                C.sub.2 H.sub.5                                                                         (S) CH(CH.sub.3)C.sub.6 H.sub.5                                                              2,000                                                C.sub.2 H.sub.5                                                                         (R) CH(CH.sub.2 OCH.sub.3)C.sub.6 H.sub.5                                                    4,000                                                (CH.sub.3).sub.2 CH                                                                     (R) CH(CH.sub.2 OCH.sub.3)C.sub.6 H.sub.5                                                    4,000                                                ______________________________________                                         *The sweetening potency is given on a weight basis compared to a 2%           solution of sucrose.                                                     

We claim:
 1. A compound of formula: ##STR15## in which: Y is C₂ H₅,CH(CH₃)₂ or (R) CH(OH)CH₃ ; andR" is (S) CH(C₂ H₅)C₆ H₅, (S) CH(CH₃)C₆H₅ or (R) CH(CH₂ OCH₃)C₆ H₅.
 2. The compound according to claim 1characterized in that it is theN-(3,3-dimethylbutyl)-L-aspartyl-D-α-aminobutyric acidN-(S)-1-phenyl-1-propan-amide of formula: ##STR16##
 3. The compoundaccording to claim 1 characterized in that it is theN-(3,3-dimethylbutyl)-L-aspartyl-D-valine N-(S)-1-phenyl-1-propanamideof formula: ##STR17##
 4. The compound according to claim 1 characterizedin that it is the N-(3,3-dimethylbutyl)-L-aspartyl-D-α-aminobutyric acidN-(S)-1-phenyl-2-meth-oxy-1-ethanamide of formula: ##STR18##
 5. Thecompound according to claim 1 characterized in that it is theN-(3,3-dimethylbutyl)-L-aspartyl-D-valineN-(S)-1-phenyl-2-methoxy-1-ethanamide of formula: ##STR19##